摘要
根据GenBank中登录的猪流行性腹泻病毒(PEDV)、传染性胃肠炎病毒(TGEV)和猪A群轮状病毒(GAR)的基因序列保守区,分别设计了3对引物。通过对引物浓度、退火温度等条件进行优化,建立了检测PEDV、TGEV和GAR的三重RT-PCR方法,并对其灵敏度、特异性进行了检测。该方法的最低检测量分别为PEDV 0.24pg、TGEV 2.3pg、GAR 1.5pg;特异性试验显示,它对猪瘟病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪萨佩罗病毒、猪博卡病毒、猪环曲病毒、猪源大肠杆菌、猪链球菌2型、猪源沙门菌、空肠弯曲杆菌和金黄色葡萄球菌等均不产生非特异性扩增。利用该方法对采自四川省6个猪场的46份样品进行检测,结果,PEDV阳性率达76.1%,TGEV阳性率为19.6%,而GAR未被检出;表明同场样品中存在PEDV与TGEV的混合感染。同时用文献报道的单一RT-PCR法对上述样品进行检测,结果符合率均为100%。结果表明,建立的三重RT-PCR具有较高的灵敏度和特异性,可用于PEDV、TGEV和GAR的检测。
In order to establish a multiplex RT-PCR that could simultaneously detect porcine epidemic diarrhea virus（PEDV）, transmissible gastroenteritis virus（TGEV）,and porcine group A rotavirus（GAR）, three pairs of special primers were designed according to the published sequences of PEDV, TGEV and GAR in GenBank. Three different amplicons with sizes of 681 bp,440 bp,and 274 bp for PEDV,TGEV,and GAR, respectively, were yielded by optimizing the reaction conditions. Testing of the sensitivity of RT-PCR indicated that the lowest detection limit were 0.24 pg for PEDV,2.3 pg for TGEV and 1.5 pg for GAR,respectively. For the specificity test, no amplicons were observed in all the negative controls. A total of 46 specimens from piglets with acute diarrheas were tested by triplex RT-PCR, and the positive rate was 76.1% for PEDV, 19. 6% for TGEV,0% for GAR, respectively, which were consistent with the result using routine RT-PCR. This study indicates that triplex RT-PCR is a simple assay and may be a potentially useful for rapid, sensitive, and cost-effective etiological diagnostic tool for acute viral diarrheas in piglets.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2013年第10期1047-1051,共5页
Chinese Veterinary Science
基金
"十二五"国家高技术研究发展(863)计划项目(2012AA101304)
西南民族大学中央高校博士创新项目(13NZYBS12)
作者简介
任玉鹏（1986-），男，四川雅安人，硕士，助理实验师，研究方向为动物疫病的病原分子生物学。
通信作者，副教授，主要从事动物疫病的病原分子生物学研究，E—mail：binovy_@sina．com
